Method of inducing differentiation of mesenchymal stem cells into neurons

ABSTRACT

The present invention relates to a method for inducing differentiation of bone marrow-derived mesenchymal stem cells into mature neurons by culturing them in an optimal medium supplemented with necessary composition. According to the pre-induction method of the invention and a method for inducing differentiation of mesenchymal stem cells into neurons by culturing them in neuronal induction media (NIM) containing butyl hydroxyanisole, forskolin and VPA, mesenchymal stem cells can be effectively differentiated into neurons or motor neurons, which thereby can be effectively used as a therapeutic agent for cell therapy for neurodegenerative diseases.

TECHNICAL FIELD

The present invention relates to a method for inducing differentiationof bone marrow-derived mesenchymal stem cells into mature neurons byculturing them in an optimum medium supplemented with necessarycomposition.

BACKGROUND ART

Stem cells are the cells of the pre-differentiation stage before beingdifferentiated into each tissue forming cell, indicating that they haveself-renewal capacity with unlimited proliferation potential beforebeing differentiated and at the same time have pluripotency withpotential for differentiation into various tissue cells by a specificstimulus. That is, even after repeated culture, self-renewal capacitydoes not decrease, and stem cells can be differentiated into varioustypes of cells.

Stem cells are largely divided into embryonic stem cells (ES cells) andadult stem cells according to the differentiation potential. After asperm meets an ovum, they are fertilized and developed to form ablastocyst. Embryonic stem cells are isolated from inner cell mass(ICM), which is supposed to be developed into a fetus, in the very earlystage blastocyst before the fertilized egg is implanted in theendometrium. These embryonic stem cells are pluripotent cells that areable to be differentiated into every tissue generated from 3 embryonicgerm layers (endoderm, ectoderm and mesoderm).

In the meantime, adult stem cells are organ specific stem cells that areisolated from an adult whose development has been completed or placentain which organ forming stage is actively undergoing. Potency of thoseadult stem cells is pluripotent, which means the potency is generallylimited to tissue forming cells. Adult stem cells remain in organs ofeven grown-up and thus play a role in supplementing normal orpathological cell loss. The most representative adult stem cells arehematopoietic stem cells in bone marrow and mesenchymal stem cells to bedifferentiated into connective tissue cells except blood cells.Hematopoietic stem cells are differentiated into various blood cellssuch as erythrocytes and leucocytes and mesenchymal stem cells aredifferentiated into osteoblasts, chondroblasts, adipocytes andmyoblasts.

Recently human embryonic stem cells were successfully isolated and theclinical application thereof has been a major concern. The best interestof stem cell application is the use as a perfect cell supplier for cellreplacement therapy. Neurodegenerative diseases such as Parkinson'sdisease and Alzheimer's disease, quadriplegia caused by spinal cordinjury, leukemia, stroke, juvenile diabetes, myocardial infarction andliver cirrhosis are caused by the destruction of cells forming tissueand permanent functional disorder. To supplement cells to make up thelack of cells caused by cell destruction or malfunction, cellreplacement therapy has been proposed.

Even though cell replacement therapy has been confirmed to haveastonishing effect, it still has limitation for clinical application.The conventional method to supply cells is that fully differentiatedcells are isolated from a donor and then transplanted to a patient. Butit is very difficult to obtain cells enough for a patient. To solve theproblem of short cell supply, both embryonic stem cells and adult stemcells can be isolated, proliferated and differentiated in vitro into aspecific cell for cell replacement therapy.

However, due to their excellent self-renewal capacity, embryonic stemcells might induce teratoma and the proliferation of undifferentiatedcells when they are transplanted in a living body for cell therapy.Since the efficiency in differentiation into specific target cells ofembryonic stem cells is low, it might cause side effects by the cellblend with other non-targeted differentiated cells when they aretransplanted in a patient. Therefore, more elaborate method of inducingdifferentiation is required for safer clinical application of embryonicstem cells.

In the meantime, cell replacement therapy using adult stem cells hasalso problems that cell proliferation is reduced under long-termculture; and/or differentiation potency might be modified so thatdifferentiation into unwanted cells occurs. Neurodegenerative diseasesuch as Parkinson's disease can be treated by neuron transplantation.But, it is very difficult to obtain neural stem cells directly from apatient. Thus, neural stem cells isolated from the fetal brain have beenproliferated and differentiated into neurons in vitro for treatment.However, to treat one patient, generally two fetus brains are required,which causes ethical issues in addition to the shortage in supply.Moreover, most neural stem cells are differentiated into astrocytes invitro rather than into neurons, and they can induce immune rejection.

If it is possible to differentiate bone marrow-derived mesenchymal stemcells into neurons, it will solve the problems of a short cell supplyand immune rejection since autologous bone marrow is used. It has been acommon belief so far that a kind of stem cells is differentiated onlyinto a specific tissue cell belonging to the same lineage. Mesenchymalstem cells are able to form in vitro colonies in the presence of variousgrowth factors such as platelet-derived growth factor, basic fibroblastgrowth factor, TGF-β (transforming growth factor-β) or EGF (Kuznetsov etal., Br. J. Haematol. 97:561, 1997; van den Bos C et al., Human Cell10:45, 1997). Approximately ⅓ of early adherent cells have pluripotency,so that they can be differentiated into connective tissue cells such asosteoblasts, chondroblasts and adipocytes (Pittenger MF et al., Science284:143, 1999). In addition, Ferrari et al reported previously that bonemarrow is the source of myogenic precursor cells involved in theformation of new muscles (Ferrari G et al., Science 279:1528, 1998).

Recent reports say that mesenchymal stem cells used to be known to bedifferentiated only into connective tissues are differentiated intonerve cells, too. For example, Sanchez-Ramos et al reported that whenmesenchymal stem cells were cultured in the presence of retinoic acidand BNDF (brain-derived neurotrophic factor), the cells weredifferentiated into neurons and astrocytes (Sanchez-Ramos et al., Exp.Neurology 164:247-256, 2000). In the meantime, Dale Woodbury et alreported that bone marrow-derived mesenchymal stem cells could bedifferentiated into neurons when they were cultured in the presence ofantioxidants such as β-mercaptoethanol or DMSO (dimethyl sulfoxide)(Dale Woodbury et al., J. Neuro. Res. 61:364-370, 2000).

However, induction of differentiation of mesenchymal stem cells intoneurons is still limited. First, a growth factor should bind to aspecific growth factor receptor expressed endogenously in the cell forintracellular signal transmission. But, there has been no report aboutthe expression of such growth factor receptor in mesenchymal stem cells,yet. Unless the expression of such a growth factor receptor is clearlydetected, the concentration of a growth factor to activate a receptorcannot be determined. If a growth factor does not bind to a receptorunder natural physiological environment (37° C.), it will be hydrolyzedvery fast by various enzymes. Thus, the activity of such growth factorwill vary in a medium. Therefore, many scientists have tried to inducedifferentiation of mesenchymal stem cells into neurons by using aninduction medium containing antioxidants such as DMSO and BHA instead ofa growth factor. However, the results were not satisfactory.Particularly, the differentiation of mesenchymal stem cells into neuronscould be possible using DMEM supplemented with various compounds(Bertani N et al., J Cell Sci, 118, 3925-3936, 2005; Woodbury D et al.,J Neurosci Res, 69, 908-917; Guillermo M-E et al., 21, 437-448, 2003),but reproducibility of the result was very low and the marker ofdifferentiated neurons could not be detected (Bertani N, J Cell Sci,118, 3925-3936). Zhao et al tried to induce stable differentiation ofmesenchymal stem cells into neurons by adopting two-phase inductionmethod (Zhao et al., Exp Neurol, 190, 396-406, 2004). Compared with theearlier trials, this attempt resulted in better differentiation, but thedifferentiation induction time was extended to 24 hours and the markerexpression was not enough to confirm the differentiation of mesenchymalstem cells into mature neurons.

Therefore, the present inventors introduced two-phase pre-inductionsystem and tried to optimize the condition of the neuronal inductionmedium. The present inventors also finally completed this invention byconfirming the expression of a marker for differentiated mature neuronsand reproducible differentiation of mesenchymal stem cells into neurons.

DISCLOSURE Technical Problem

It is an object of the present invention to provide neurons useful forcell therapy by inducing reproducible differentiation of mesenchymalstem cells into neurons by using an optimized induction medium and aculture method.

Technical Solution

To achieve the above object, the present invention provides a method forinducing differentiation of mesenchymal stem cells into neurons,comprising the following steps:

1) Performing pre-induction of mesenchymal stem cells twice; and

2) Inducing differentiation of the pre-differentiated mesenchymal stemcells of step 1) in a neuronal induction medium containing butylatedhydroxyansiole (BHA), forskolin and valproic acid (VPA) for 2˜8 hours.

The present invention also provides a neuronal induction mediumcontaining butylated hydroxyansiole (BHA), forskolin and valproic acid(VPA) to induce differentiation of pre-differentiated mesenchymal stemcells into neurons.

The mesenchymal stem cells hereinabove is preferably isolated from humanbone marrow. Particularly, mononuclear cells are isolated from bonemarrow, which are cultured for 1˜2 weeks. Then, differentiation-readyhematopoietic stem cells are all differentiated to generate mature bloodcells and remaining stem cells are isolated, by which mesenchymal stemcells are obtained. In addition to the separation of mesenchymal stemcells from mononuclear cells isolated from bone marrow, the wholemononuclear cells containing mesenchymal stem cells can be culturedaccording to the method of the present invention to mass-produceneurons.

The method of inducing differentiation of mesenchymal stem cells intoneurons is that pre-induction of step 1) is performed twice withincreasing the concentration of β-mercaptoethanol and thendifferentiation is induced in a neuronal induction medium containing100˜200 μM of BHA, 9˜11 μm of forskolin and 1.5˜2.5 mM of VPA. Duringthe pre-induction, it is preferred to increase the concentration ofβ-mercaptoethanol for the second pre-induction up to 1.5˜2 fold, morepreferably 2 fold, from the concentration for the first pre-induction.The second pre-induction time is preferably shortened to ¼˜⅛ of thefirst pre-induction time and ⅛ time is preferred. The neuronal inductionmedium preferably contains 200 μM of BHA, 10 μm of forskolin and 2 mM ofVPA and induction time is preferably 2˜8 hours and more preferably 3˜5hours (see FIG. 1 and FIG. 2). RT-PCR was performed to find that aneuronal marker expression was the highest in the group treated with NIMfor 4 hours, whereas the immunofluorescent staining to detect a specificprotein expression in each cell confirmed that NF-M expression wasdominant in the group treated with NIM for 6 hours (see FIG. 5).

The butylated hydroxyanisole (BHA) is an organic compound comprising twoisomers, 2-tert-butyl-4-hydroxyanisole and3-tert-butyl-4-hydroxyanisole, and has the characteristics of anantioxidant. BHA is known to inhibit an intracellular signal pathwayregulated by reactive oxygen intermediates, such as nuclear factor(NF)-kB activation (Sasada T et al., J. Clin. Invest., 97, 2268-2276,1996). Thus, such an antioxidant activity leads to the increase inneuroprotection (Poeggeler B et al., J. Neurochem, 95, 962-973, 2005).Forskolin (7 beta-acetoxy-8, 13-epoxy-1 alpha, 6 beta, 9alpha-trihydroxy-labd-14-ene-11-one) activates adenylcyclase andincreases cyclic AMP (cAMP) to stimulate intracellular signalingpathway. cAMP is a crucial signal transmitter which is necessary forcell response and also plays an important role in activation of proteinexpression for cell survival and differentiation. Valproic acid (VPA)inhibits histone deacetylase (HDAC) activity directly and thisinhibition suppresses cell growth, which leads to increases in thedifferentiation of tumor cells. VPA also activates signal transductionpathway such as extracellular signal-regulated kinase (ERK) pathway toincrease cell survival (Hsieh J et al., PNAS, 101, 16659-16664, 2004).It is also reported that ERK is involved in the proliferation ofdifferentiated cells (Joneson T et al., J Biol Chem, 273, 7743-7748,1998; Christerson LB et al., Cell Motil Cytoskeleton, 43, 186-198, 1999;Stariha RL & Kim SU, Microsc Res Tech, 52, 680-688, 2001; Vaudry D etal., J Neurochem, 83, 1272-1284, 2002).

In general, the concentration of DMSO to fast-freeze cells is 10%.However, in the present invention, 2% DMSO was used only for 6 hours, sothat cytotoxicity might not be induced. In the present invention,mesenchymal stem cells were isolated from human bone marrow and culturedin a medium designed to efficiently induce differentiation ofmesenchymal stem cells into neurons in vitro, suggesting that there wasno need to use this medium directly in human. That is, only cellsdifferentiated in the medium can be isolated and injected into humanbody for therapy.

The present inventors re-conducted the earlier experiments to inducedifferentiation of mesenchymal stem cells into neurons (Zhao et al., ExpNeurol, 190, 396-406, 2004; Bertani N et al., J Cell Sci, 118,3925-3936, 2005; Woodbury D et al., J Neurosci Res, 69, 908-917;Guillermo M-E et al., 21, 437-448, 2003). However, the result was notconsistent with that of the earlier experiment. The efficiency ofneuronal induction was very low, and marker expression was hardlydetected, which means the differentiation of mesenchymal stem cells intoneurons was difficult to be confirmed (see FIG. 7). In addition, thedifferentiation of mesenchymal stem cells into neurons was not enoughand unsatisfactory (see FIG. 6). Thus, the present inventors developed ahighly reproducible novel method for inducing differentiation ofmesenchymal stem cells into neurons by eliminating unnecessary compoundsfor neuronal induction. Then, the expression of differentiated neuronalmarkers (neurofilament 150 kDa, NF-M; neurofilament 68 kDa, NF-L),including a motor neuron maker (Islet-1) was confirmed by RT-PCR (seeFIG. 3). The present inventors further confirmed the expression of NF-Mprotein in the differentiated neurons by immunocytochemistry method (seeFIG. 4). In addition, the inventors observed that the combination of themethod of Zhao et al and the method of Bertani et al induceddifferentiation of mesenchymal stem cells into neurons more efficientlythan the method of Zhao et al was used alone. Furthermore, expression ofthe marker for differentiated neuron was increased and this result washighly reproducible (see FIG. 9).

DESCRIPTION OF DRAWINGS

The application of the preferred embodiments of the present invention isbest understood with reference to the accompanying drawings, wherein:

FIG. 1 is a set of photographs illustrating the morphological changesover the times of treating NIM (neuronal induction media) to mesenchymalstem cells,

FIG. 2 is a set of photographs illustrating the expression levels ofneuronal markers over the times of treating NIM (neuronal inductionmedia) to mesenchymal stem cells,

FIG. 3 is a set of photographs illustrating the gene expressions of theNIM non-treated control group and the NIM-treated group,

FIG. 4 is a set of photographs illustrating the expressions of neuronalmarkers over NIM treating times (0, 3, 6, 9 and 24 hr), confirmed byimmunofluorescence assay,

Red: nestin Green: NF-M Blue: DAPI

FIG. 5 is a set of photographs illustrating the expressions of neuronalmarkers over NIM treating times (2, 4, 6 and 8 hr), confirmed byimmunofluorescence assay,

Red: NF-M Green: nestin

FIG. 6 is a set of microphotographs illustrating that differentiation ofmesenchymal stem cells into neurons was induced by the method of Zhao etal, but the differentiation of mesenchymal stem cells into neurons wasnot successful,

FIG. 7 is a set of photographs illustrating the comparison of the geneexpressions among the NIM-treated mesenchymal stem cell group, amesenchymal stem cell group treated by the method of Zhao et al and theNIM non-treated group,

FIG. 8 is a set of microphotographs illustrating that differentiation ofmesenchymal stem cells into neurons was comparatively clear when themethod of Zhao et al was used along with the method of Bertani et al,compared with when the method of Zhao et al was used alone,

FIG. 9 is a set of photographs illustrating the comparison of geneexpressions between the mesenchymal stem cell group treated by thecombination of the method of Zhao et al and the method of Bertani et aland the non-treated group.

MODE FOR INVENTION

Practical and presently preferred embodiments of the present inventionare illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, onconsideration of this disclosure, may make modifications andimprovements within the spirit and scope of the present invention.

EXPERIMENTAL EXAMPLE 1 Recurrence of Differentiation of Mesenchymal StemCells into Neurons by the Method of the Present Invention

<1-1> Human Mesenchymal Stem Cell (hMSC) Culture

Poietics Normal Human Mesenchymal Stem Cells were purchased fromCambrex, USA. The above stem cells were subcultured two times in a MSCgrowth medium (MSCGM-500 ml of mesenchymal cell growth supplement, 10 mlof 200 mM L-glutamine, 0.5 ml of penicillin-streptomycin, Cambrex, USA)comprising a basic medium and a growth supplement, and then transferredinto DMEM (Gibco, USA) supplemented with 10% FBS (Gibco, USA), 100 ng/mlpenicillin and 100 U/ml streptomycin, followed by further culture in a37° C., 5% CO₂ incubator. After three days of culture in the incubator,the medium was removed and the cells were washed with phosphate-bufferedsaline (PBS) to completely remove the remaining medium. Cells weredetached with 0.1% trypsin/EDTA (Gibco, USA) and then diluted with a newmedium at the ratio of 1:3, followed by subculture.

<1-2> Induction of Neuronal Differentiation of hMSC

Mesenchymal stem cells were subcultured in DMEM supplemented with 10%FBS and penicillin-streptomycin. To induce neuronal differentiation, thestem cells were cultured in a general medium containing 1 mMβ-mercaptoethanol (Sigma, USA) for 24 hours and then the medium wasreplaced with another general medium containing 2 mM β-mercaptoethanol,followed by further culture for 3 hours (pre-induction). After thepre-induction, the medium was replaced once again with a neuronalinduction medium (NIM) prepared by adding 2% DMSO (Sigma, USA), 200 μMbutylated hydroxyanisole (Sigma, USA), 10 μM forskolin (Sigma, USA), 2mM valproic acid (Sigma, USA) and 10 mM potassium chloride (Sigma, USA)to the DMEM supplemented with N2 supplements (Gibco, USA) instead of 10%FBS. After culture for a required times (0, 2, 4, and 8 hours), thechanges of morphology were observed under the microscope.

From the observation of morphology of the above experimental group wasconfirmed that the mesenchymal stem cells cultured in the NIM changedinto neuron-like morphology (FIG. 1).

<1-3> Detection of Neuronal Differentiation Markers by RT-PCR

RNA was extracted from both non-treated control mesenchymal stem cellsand NIM-treated mesenchymal stem cells by using trizole (Invitrogen,USA). Reverse transcription (RT) was performed with 2 μg of theextracted RNA by using MMLV reverse transcriptase (MMLV RTase; Promega,USA). Particularly, RT was performed with 50 μl of volume by using 0.5μg of oligo (dT) primer, 2.5 mm dNTPs, 5× MMLV buffer, RNase inhibitorand MMLV RTase. Then, PCR amplification was performed using a PCRmachine (Bio-Rad, USA) as follows; predenaturation at 94° C. for 5minutes, denaturation at 94° C. for 45 seconds, annealing at 55˜65° C.for 45 seconds, polymerization at 72° C. for 45 seconds, 35 cycles fromdenaturation to polymerization, and final extension at 74° C. for 7minutes.

PCR was performed with the RT-product (3˜5 μl), using the primer setpresented in Table 1. For the negative control, PCR was performed withwater instead of the RT product. To increase accuracy of PCR result andequal distribution, a master mix composed of the primer set, 10× buffer,Taq polymerase, and 2.5 mM dNTPs was loaded in each reaction tube, towhich RT product or water was added as a template, followed by PCR. PCRwas performed as follows; predenaturation at 95° C. for 5 minutes,denaturation at 95° C. for 45 seconds, annealing at 65° C. for 45seconds, polymerization at 72° C. for 45 seconds, 35 cycles fromdenaturation to polymerization, and final extension at 72° C. for 5minutes, followed by cooling at 4° C. PCR product was electrophoresed on1.5% agarose gel, and the band size was measured by using atransilluminator.

Table 1: PCR primer set

TABLE 1 Name Sequence Size Human Nestin Forward CTCTGACCTGTCAGAAGAAT 316bp (SEQ. ID. NO: 1) Reverse GACGCTGACACTTACAGAAT (SEQ. ID. NO: 2) Humanb- Forward ATGAGGGAGATCGTGCACA 267 bp tubulin (SEQ. ID. NO: 3) III(TuJ1) Reverse CCCCTGAGCGGACACTGT (SEQ. ID. NO: 4) Human NF-M ForwardTGGGAAATGGCTCGTCATTTG 333 bp (SEQ. ID. NO: 5) ReverseCTTCATGGAAACGGCCAATTC (SEQ. ID. NO: 6) Human NF-L ForwardTCCTACTACACCAGCCATGTC 285 bp (SEQ. ID. NO: 7) ReverseTCCCCAGCACCTTCAACTTTC (SEQ. ID. NO: 8) Human beta- ForwardCCACGAAACTACCTTCAACTCC 285 bp actin (SEQ. ID. NO: 9) ReverseTCATACTCCTGCTGCTTGCTGA TCC (SEQ. ID. NO: 10)

As a result, the expressions of NF-M and NF-L, mature neuronal markers,depended on the induction time by NIM in hMCS. Particularly, theexpressions of NF-M and MF-L were hardly detected in the NIM-non-treatedcontrol group. After treating hMSC with NIM, the expressions of NF-M andNF-L were gradually increased over the times for 6 hours, which was theturning point to start decreasing of the levels (FIG. 2). Morespecifically, NIM treatment of hMSC for 4 hours highly induceddifferentiation of mesenchymal stem cells into neurons. Considering thatmature neuronal marker expression was the highest, induction time waspreferably determined to be 4 hours for efficient induction. Even thoughthe primary neuronal marker expression of the NIM-treated group wassimilar to that of the non-treated group, the expressions of matureneuronal markers (NF-M and NF-L) and motor neuronal marker (Islet-1)were clearly increased (FIG. 3). In conclusion, the method for inducingdifferentiation of the present invention induced the differentiation ofmesenchymal stem cells into neurons and further exhibited the potentialfor inducing differentiation into motor neurons.

<1-4> Detection of Neuronal Differentiation Markers byImmunofluorescence Cytochemistry Method

A cover slip (Fisher Scientific, USA) was coated with 20 μg/ml of PDL(Sigma, USA) for a day, which was then re-coated with 10 μg/ml oflaminin (Sigma, USA) for three hours, followed by washing with distilledwater 5 times. The growing mesenchymal stem cells were detached from theculture vessel using 0.1% trypsin/EDTA, which were seeded on theprepared PDL-laminin coated cover slip and then cultured in DMEM(containing 1000 mg/l glucose, WelGENE, Korea) supplemented with 10% FBSfor a day. Non-treated control mesenchymal stem cells or NIM-treatedstem cells of Example <1-2> were treated with 4% (v/v) paraformaldehydeat room temperature for 30 minutes, followed by washing with 1× PBSthree times. Then, the cells were treated with 0.2% triton X100 for 10minutes, permeated and washed with 1× PBS three times. To avoidnon-specific binding, blocking buffer (1× PBS containing 5% goat serum)was treated thereto for one hour. The primary antibody (Table 2) dilutedin the blocking buffer at 4° C. was bound to the mesenchymal stem cellsovernight, followed by washing with 1× PBS three times. Then,FITC(fluorescein isothiocyanate)-binding or Cy3-binding-anti-mounse oranti rabbit (Jackson Immunoresearch, USA) secondary antibody diluted inthe blocking buffer (1:500) was bound to the stem cells for one hour atroom temperature in a dark room. The nuclei were stained with DAPI(Santa Cruz, USA) diluted in PBS (1:2,500) for 10 minutes, washed with1× PBS three times and then photographed by a confocal microscope(FluoView™ Confocal Microscope, Olympus, Japan), followed byhistological analysis.

As a result, the stem cell marker nestin was expressed at Oh but theexpression was reduced over the NIM treating times. The expressions ofneuronal markers, such as NeuN, MAP-2 and NF-M (neurofilament 150 kDa)were increased over the NIM treating times and their morphology was morelike neuron-like cells, suggesting that the stem cells were beingdifferentiated into neurons (FIG. 4).

The expressions of neuronal marker proteins were detected over the NIMtreating times (2, 4, 6 and 8 hours) by immunofluorescence. As a result,NF-M expression was increased in NIM 6 group (cells were treated withNIM for 6 hours), whereas nestin expression was reduced therein (FIG.5).

TABLE 2 Table 2: Dilution ratios of primary antibodies Name Dilutionratio Host Nestin 1:300 Mouse NF-M 1:400 Rabbit NeuN 1:500 Mouse MAP21:200 Mouse

COMPARATIVE EXAMPLE 1 Recurrence of Differentiation of Mesenchymal StemCells into Neurons by the Method of Zhao et al

<1-1> Induction of Differentiation of hMSC into Neurons

The present inventors induced differentiation of hMSC into neuronsaccording to the method of Zhao et al (Zhao et al., Exp Neurol, 190,396-406, 2004). The mesenchymal stem cells prepared in ExperimentalExample <1-1> were cultured in a general medium (DMEM/F-12 containing10% FBS and penicillin-streptomycin) and then transferred into anothermedium supplemented with 1 mM β-mercaptoethanol (Sigma, USA) andcultured for 24 hours to induce neuronal differentiation. The medium wasreplaced with another general medium supplemented with 2 mMβ-mercaptoethanol and the stem cells were further cultured for 3 hours,leading to the pre-induction. The medium was replaced again with aneuronal induction medium (NIM) prepared by adding 2% DMSO (Sigma, USA)and 200 μM butylated hydroxyanisole (Sigma, USA) to DMEM supplementedwith N2 supplements (Gibco, USA) instead of 10% FBS, followed by furtherculture for one˜two more hours. The changes of morphology were observedunder a microscope.

As a result, the differentiation of mesenchymal stem cells into neuronswas not confirmed by morphological changes, suggesting that therecurrence of differentiation was not successful (FIG. 6).

<1-2> Detection of Neuronal Differentiation Markers by RT-PCR

The method to extract RNA from non-treated control mesenchymal stemcells and NIM-treated mesenchymal stem cells of Comparative Example<1-1> and the method to detect a band using electrophoresis andtransilluminator after reverse transcription and PCR were the same asdescribed in Experimental Example <1-3>.

As a result, the level of neuronal marker was not increased by thedifferentiation of mesenchymal stem cells into neurons according to theprocedure of Comparative Example <1-1> (FIG. 7). This result indicatesthat differentiation induction by the method of Zhao et al was not asefficient as the method of the present invention.

COMPARATIVE EXAMPLE 2 Recurrence of Differentiation of Mesenchymal StemCells into Neurons by the Combination of the Method of Zhao et al andthe Method of Bertani et al

<2-1> Induction of Differentiation of hMSC into Neurons

The present inventors induced differentiation of hMSC into neurons bythe combination of the method of Zhao et al (Zhao et al., Exp Neurol,190, 396-406, 2004) and the method of Bertani et al (Bertani N et al., JCell Sci, 118, 3925-3936, 2005). The mesenchymal stem cells prepared inExperimental Example <1-1> were cultured in a general medium (DMEMcontaining 10% FBS and penicillin-streptomycin) and then transferredinto another general medium supplemented with 1 mM β-mercaptoethanol(Sigma, USA), which were cultured for 24 hours to induce neuronaldifferentiation. The medium was replaced with another general mediumsupplemented with 2 mM β-mercaptoethanol and the stem cells were furthercultured for 3 hours, leading to the pre-induction. The medium wasreplaced again with a neuronal induction medium (NIM) prepared by adding2% DMSO (Sigma, USA) and 200 μM butylated hydroxyanisole (Sigma, USA),10 μM forskolin (Sigma, USA), 2 mM valproic acid (Sigma, USA) and 10 mMpotassium chloride (Sigma, USA) to DMEM supplemented with N2 supplements(Gibco, USA) instead of 10% FBS, followed by further culture for arequired time (0, 3, 6, 9 hours and 1 day).

As a result, the differentiation of mesenchymal stem cells into neuronsand recurrence of the differentiation was improved, compared when themethod of Zhao et al was used alone (FIG. 9). And, morphologicalanalysis also confirmed the differentiation of mesenchymal stem cellsinto neurons (FIG. 8).

<2-2> Detection of Neuronal Differentiation Markers by RT-PCR

The method to extract RNA from non-treated control mesenchymal stemcells and NIM-treatedmesenchymal stem cells of Comparative Example <2-1>and the method to detect a band using electrophoresis andtransilluminator after reverse transcription and PCR were the same asdescribed in Experimental Example <1-3>.

As a result, the expression of the neuronal marker was detected duringthe differentiation of mesenchymal stem cells into neurons according tothe method of Comparative Example <2-1>, suggesting that this method ismore efficient in inducing differentiation of mesenchymal stem cellsinto neurons than the method of Zhao et al (FIG. 9).

INDUSTRIAL APPLICABILITY

As explained hereinbefore, the method of the present invention composedof double pre-inductions and using neuronal induction media (NIM)supplemented with butylated hydroxyanisole, forskolin and VPA canprovide neurons or motor neurons for the cell therapy ofneurodegenerative diseases by inducing differentiation of mesenchymalstem cells into neurons or motor neurons.

Those skilled in the art will appreciate that the conceptions andspecific embodiments disclosed in the foregoing description may bereadily utilized as a basis for modifying or designing other embodimentsfor carrying out the same purposes of the present invention. Thoseskilled in the art will also appreciate that such equivalent embodimentsdo not depart from the spirit and scope of the invention as set forth inthe appended claims.

1. A method for inducing differentiation of mesenchymal stem cells intoneurons, comprising the following steps: 1) Performing pre-induction ofmesenchymal stem cells twice; and 2) Inducing differentiation of thepre-differentiated mesenchymal stem cells of step 1) in a neuronalinduction medium containing butylated hydroxyansiole (BHA), forskolinand valproic acid (VPA) for 2˜8 hours.
 2. The method for inducingdifferentiation of mesenchymal stem cells into neurons according toclaim 1, wherein the content of β-mercaptoethanol added for the secondpre-induction is 1.5˜2 fold increased from the content added for thefirst pre-induction.
 3. The method for inducing differentiation ofmesenchymal stem cells into neurons according to claim 1, wherein thesecond pre-induction time is reduced to ¼˜⅛ of the first pre-inductiontime.
 4. The method for inducing differentiation of mesenchymal stemcells into neurons according to claim 1, wherein the secondpre-induction time is reduced to ⅛ of the first pre-induction time. 5.The method for inducing differentiation of mesenchymal stem cells intoneurons according to claim 1, wherein the neuronal induction mediumcontains 100˜200 μM BHA, 9˜11 μM forskolin and 1.5˜2.5 μM VPA.
 6. Themethod for inducing differentiation of mesenchymal stem cells intoneurons according to claim 1, wherein the induction was performed withthe neuronal induction medium for 2˜8 hours.
 7. A neuronal inductionmedium containing butylated hydroxyanisole (BHA), forskolin and valproicacid (VPA) to induce differentiation of pre-induced mesenchymal stemcells into neurons.
 8. The neuronal induction medium according to claim7, which contains 100˜200 μM BHA, 9˜11 μM forskolin and 1.5˜2.5 μM VPA.